Deregulated signalling by the family of epidermal growth factor receptors (EGFR) is implicated i may human cancers. This family consists of four members, EGFR and HER2 generally correlates with a poor clinical prognosis. Overexpression of HER3 has been observed in a variety of human adenocarcinomas. Recent evidence suggests that, unlike the other UGFR family members, HER4 may play a role in differentiation of some normal and transformed cells. This proposal focuses on elucidating the differentiation signal transduced through HER4 in human breast cancer cells. A breast cancer cell system will be developed in which the intracellular signalling elements triggered by HER4 can be distinguished from the elements of the proliferative pathway triggered by EGFR. To accomplish this, EGFR or an EGFR:HER4 chimera will be stably transfected into the EGFR-lacking breast cancer cell line, MDA-MB-453. Transfected cells will be stimulated with EGF and the growth-promoting actions of egfr compared to the differentiation-promoting actions of EGFR:HER4 for identification of specific HER4 substrates, adapter molecules, and downstream signalling events. Additionally, novel HER4 substrates will be isolated by the yeast two-hybrid method, an in vivo detection system of protein-protein interactions. This study will help to elucidate the signalling mechanisms involved in the HER4 differentiation pathway and lead to a better understanding of how breast cancer cells escape this normal process.